Malignant gliomas, the most common subtype of primary brain tumors, are among the most devastating, incurable brain disorders despite advances in surgery, radiotherapy, and chemotherapy. Replication-conditional viral mutants (oncolytic viruses) offer an alternative modality for treating these tumors because of their potential to destroy tumor cells selectively, spread their progeny throughout tumor tissue, and eradicate tumor cells through oncolysis and vaccination effects. However, issues still to be resolved include inefficient delivery, propagation, and spread of oncolytic viruses in tumor tissue in vivo. The use of tumor-targeting cells as delivery vehicles is a promising strategy to circumvent some limitations of conventional oncolytic virotherapy because carrier cells pre-loaded with oncolytic virus could: 1) deliver the virus to tumor cells, infiltrating beyond the reach of directly injected viruses;2) escape host immune surveillance;and 3) be further engineered to enhance antitumor efficacy. In this proposal, we will develop a herpes simplex virus (HSV) amplicon-based system that can transform virtually any adenovirus (Ad)-permissive cells into carriers of oncolytic Ads. HSV amplicon vectors are plasmid-based, replication-deficient vectors that can accommodate up to 150 kb of exogenous DNA. Taking advantage of the high-capacity vector system, the entire genome of an oncolytic Ad equipped with a regulatable gene switch will be delivered to carrier cells of interest through efficient HSV infection. The hybrid amplicon construct will be designed so that the amplicon-encoded Ad genome in infected carrier cells stays latent under normoxic conditions, but becomes activated and produces Ad progeny upon exposure to hypoxia, a hallmark of the tumor microenvironment. In this exploratory/developmental R21 project, we will focus on the development and verification of the amplicon-based Ad-producing vector system and screen candidate carrier cells that can deliver and produce oncolytic Ads. PUBLIC HEALTH RELEVANCE: Engineered viruses can be used as tumor-targeting agents to treat human malignancies. We propose to develop a new methodology to convert any cells of interest into vehicles that selectively target malignant cells with tumor-killing viruses. The work may provide a technical foundation for a novel strategy for treating brain tumors and other human malignancies.